GenieClone 5 Minute Ligation mix is a ready-to-use 2 × premix solution suitable for sticky-end ligation, blunt-end DNA ligation and TA ligation. The kit contains an optimised T4 DNA ligase that catalyzes the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini in double-strand DNA or RNA. The unique buffer system comes with optimized ligation enhancer making the reaction much more efficient and convenient. Ligation can be completed in 5 min at 25℃. The GenieClone 5 Minute Rapid Ligation mix can be used directly to transform many types of chemically competent cells.
Store at -30 ~ -15℃. Transportation conditions: ≤0℃
• Sticky end ligation
• Blunt end ligation
• TA ligation
• Linker or Adapter ligation
Store at -30 ~ -15℃. Transportation conditions: ≤0℃.
Summary of the Experimental Process
A: Sticky end ligation
B: Blunt end ligation
C: TA ligation
Figure 1: Process summary of GenieClone 5 Minute Ligation mix
1. Ligation Reaction
1) Prepare the reaction mix on ice:
0.03 – 0.3 pmol
2 x Universal Ligation Mix
To 10 µl
The optimal vector usage (0.03 pmol) = [0.02 × base pair of vector] ng
The optimal fragment usage (0.09 pmol) = [0.06 × base pair of fragment] ng
For example, when a 1 kb fragment is cloned into a 4 kb cloning vector, the optimal usage of cloning vector: 0.02 × 4,000 = 80ng; the optimal usage of fragment: 0.06 × 1,000 = 60 ng.
• The molar ratio of vector: fragment ranged from 1:1 to 1:10, and the optimal molar ratio was 1:3.
• The optimal input of vector is 20 - 100 ng; When the calculated optimal amount of DNA exceeds this range, the lowest/highest amount can be directly selected.
• When a blunt-end vector is ligated to an fragment, the vector should be dephosphorylated to prevent own cyclization.
• Nanodrop, Onedrop and Qubit are recommended for concentration measurement.
2) Mix thoroughly by pipetting up and down several times (do not vortex), and centrifuge briefly to collect the reaction liquid.
3) Incubate 5 min at 25℃; Cool down to 4℃ or place on ice immediately.
• Other conditions for ligation: overnight at 4℃ or 16℃.
• When the total volume of vector and fragment is more than 5 μl, the reaction system can be enlarged to 20 μl. For blunt-end ligation or TA ligation, the reaction time can be increased to 2 h to improve the efficiency.
• The product need to be purified by spin column or chloroform extraction before electro-transformation.
• The product can be stored for one week at -20℃.
2. Transformation Protocol
1) Chemically competent cells for cloning are thawed on ice (e.g. Fast-T1 competent cells)
2) Add 5 - 10 μl of the ligation product into 100 μl of competent cells, and mix by fingerflicking (do not vortex the tube), then place the tube on ice for 30 min.
• The transformed volume of the ligation product should not exceed 1/10 of the volume of competent cells.
3) Heat shock at 42℃ for 30 sec, then return the tube to ice for 2 - 3 min immediately.
4) Add 900 μl of SOC or LB liquid medium (without antibiotics), and incubate for 1 h at 37℃ with rotation or shaking (200 - 250 rpm).
5) Warm selection plates at 37℃.
6) Centrifuge at 5,000 rpm for 5 min and discard 900 μl supernatant. Resuspend the bacteria and coat onto plates.
7) Incubate overnight at 37℃.
3. Identification of positive clones
PCR identification; Digestion identification; Plasmid identification; DNA Sequencing.